The internal anal sphincter (IAS) plays a key role in continence and defecation. Its disorders underlie many clinical abnormalities, such as fecal incontinence and constipation. Disorders of the IAS may also be involved in a number of anal diseases such as hemorrhoids and anal fissures. Also, since disorders of the IAS occur more frequently in the elderly, studies on IAS function are of particular importance in this age group. The objectives of the present research proposal are to investigate: 1) the nature of inhibitory neurotransmitters and mediators responsible for IAS relaxation in response to the rectoanal inhibitory reflex; 2) the neural pathways in the rectoanal inhibitory reflex and role of regulatory peptides and other neurohumoral substances on IAS function; and 3) the cellular bases underlying myogenic tone in the IAS. Studies are planned in vivo and in vitro in which the activity of the IAS will be isolated from that of the external anal sphincter (EAS) by simultaneous electrical and mechanical recordings. Studies will be performed to examine the structure-activity-relationship, receptor binding, second messengers, immunocytochemical localization of physiologically relevant neuropeptides, radioimmunoassay (RIA) of neuropeptides, the direct release of mediators, membrane potential changes using intracellular electrical recordings, changes in the free intracellular Ca2+, PI turnover and protein kinase C. While these studies will yield a diverse spectrum of information, they will be specifically aimed at elucidating various aspects of the objectives mentioned above: inhibitory neurotransmitters, neurohumoral factors and neural pathways, and the cellular bases of myogenic tone. These studies will be performed in opossums, but other animal species will also be used to determine species differences in the control of the sphincter. Some studies are planned in awake animals to understand the normal physiology of the IAS. It is envisaged that the proposed studies provide important information on the physiology and pharmacology of the IAS leading to a better understanding of the pathophysiology and in turn the treatment, of anorectal disorders.